A method for detecting the content of d-glycerophosphocholine in l-alpha-glycerophosphocholine and application thereof

Abstract

The application belongs to the technical field of chemical analysis, and particularly relates to a detection method for D-glycophosphocholine content in L-alpha-glycophosphocholine and application. The detection method selects a silica gel surface polysaccharide covalent bonding cellulose type or silica gel surface polysaccharide coating cellulose type normal phase chromatographic column and an evaporative light scattering detector (ELSD), detects through an HPLC-ELSD method, has the advantages of simple operation, high sensitivity, good method specificity and reproducibility, is a high-efficiency liquid phase analysis method convenient for standardized operation, can completely separate L-alpha-glycophosphocholine from low-limit D-glycophosphocholine isomer impurities, and can accurately quantify.

Description

Technical Field

[0001] This invention belongs to the field of chemical analysis technology, specifically relating to a method and application for detecting the content of D-glucosamine in L-α-glucosamine. Background Technology

[0002] L-α-glycerophosphoryl choline (L-α-GPC) is a choline-based substance. It is a naturally occurring phospholipid metabolite and a source of choline for the synthesis of acetylcholine and phosphoric acid choline, possessing significant nutritional and health benefits. It is now listed as a dietary supplement in the United States Pharmacopeia (USP) 42-NF38. L-α-glycerophosphoryl choline has been shown to improve cognitive function, and many reports even suggest it can repair early-stage Alzheimer's disease.

[0003] Currently, L-α-glucosinolate has two sources: one is natural extraction, but this has the problems of low purity and high cost of the isolated product; the other is chemical synthesis, which not only has a wide range of raw material sources but also high purity. The chemical structure and related physical properties of high-purity L-α-glucosinolate are more beneficial to protecting the metabolism of brain tissue.

[0004] Based on existing synthetic routes and the structure of L-α-glucosinolate (L-α-glucosinolate), it is known that L-α-glucosinolate contains a chiral center, theoretically leading to the existence of an enantiomer, D-glucosinolate. In previous synthesis processes, the purity of L-α-glucosinolate was achieved by controlling the specific rotation of the product. However, specific rotation determination is susceptible to environmental influences, has low accuracy, and a wide standard range, making it impossible to accurately quantify the low-limit D-glucosinolate isomer in high-purity L-α-glucosinolate, thus failing to guide the purification and refining of L-α-glucosinolate products. Therefore, the art seeks to develop an effective and efficient method for analyzing D-glucosinolate isomers to accurately quantify the low-limit D-glucosinolate isomer in high-purity L-α-glucosinolate, thereby guiding and supporting further purification of high-purity L-α-glucosinolate.

[0005] Studies show that neither L-α-glycophosphocholine nor D-phosphatidylcholine contains chromogenic groups, making both substances undetectable by UV detectors. Furthermore, the content of D-glycophosphocholine isomers in high-purity L-α-glycophosphocholine is far lower than the content of the material itself (≤0.05%), rendering general-purpose differential detectors ineffective. L-α-glycophosphocholine and D-glycophosphocholine are chiral isomers with similar polarities, making complete and effective separation impossible with ordinary silica gel, octadecylsilane-bonded, and octylsilane-bonded columns. Therefore, current methods for controlling D-glycophosphocholine isomers in high-purity L-α-glycophosphocholine require multiple detection techniques, each time-consuming, costly, and unable to accurately quantify low-limit D-glycophosphocholine isomers. Therefore, developing an efficient method for detecting D-glucosine isomer impurities in L-α-glucosine has become an urgent issue to be addressed in this field. Summary of the Invention

[0006] The purpose of this invention is to provide a method for detecting the content of D-glucosylcholine isomer impurities in L-α-glucosylcholine based on HPLC-ELSD. The detection method can achieve rapid and accurate determination of the content of low-limit D-glucosylcholine isomer impurities in L-α-glucosylcholine, and has the advantages of simple operation, readily available reagents, high separation, and high accuracy, sensitivity and reproducibility.

[0007] The second objective of this invention is to provide an application of the method for detecting the content of D-glucosamine in L-α-glucosamine.

[0008] To solve the above-mentioned technical problems, the present invention provides a method for detecting the content of D-glucosamine in L-α-glucosamine, the method comprising the step of performing HPLC-ELSD detection on the L-α-glucosamine test solution;

[0009] The chromatographic conditions in the HPLC-ELSD detection step include:

[0010] The analytical column was a chiral normal-phase column of cellulose covalently bonded to a silica gel surface with polysaccharides or a chiral normal-phase column of cellulose coated with silica gel with polysaccharides.

[0011] The mobile phase A is an alkane solution containing 0.1-0.3% v / v diethylamine or triethylamine;

[0012] Mobile phase B is an alcoholic solution containing 0.1-0.3% v / v diethylamine or triethylamine;

[0013] The mobile phase A and mobile phase B are eluted isocratically at a volume ratio of (50-75):(50-25).

[0014] Specifically, the method for detecting the D-glucosamine content in L-α-glucosamine is as follows:

[0015] In the mobile phase A, the alkanes in the alkane solution include n-hexane, n-heptane, or isooctane;

[0016] And / or, in the mobile phase B, the alcohol in the alcohol solution includes isopropanol, anhydrous ethanol, or anhydrous ethanol containing 3-8% v / v methanol and 3-8% v / v isopropanol.

[0017] Preferably, the mobile phase A is a hexane solution containing 0.1% v / v diethylamine;

[0018] Preferably, mobile phase B is anhydrous ethanol containing 0.1% v / v diethylamine (containing 5% v / v isopropanol and 5% v / v methanol).

[0019] Preferably, in the elution process, the volume ratio of mobile phase A to mobile phase B is 7:3 for isocratic elution (v / v).

[0020] Specifically, in the method for detecting the D-glucosamine content in L-α-glucosamine, the chromatographic conditions in the HPLC-ELSD detection step further include:

[0021] The flow rate is 0.5-1.0 ml / min;

[0022] And / or, the column temperature is 25-45℃;

[0023] And / or, the injection volume is 20-50 μl;

[0024] And / or, the detection time is 40-60 minutes.

[0025] The test solution is preferably run for 50-60 minutes, while the reference solution is preferably run for 45 minutes.

[0026] Preferably, in the HPLC-ELSD detection step, the flow rate is 0.7 ml / min;

[0027] Preferably, in the HPLC-ELSD detection step, the column temperature is 30°C;

[0028] Preferably, in the HPLC-ELSD detection step, the injection volume is 30 μl.

[0029] Specifically, in the HPLC-ELSD detection step, the settings parameters of the evaporative light scattering detector include:

[0030] The drift tube temperature is 70-100℃;

[0031] And / or, the carrier gas includes high-purity nitrogen or air;

[0032] And / or, the carrier gas flow rate is 1.5-3.0 L / min;

[0033] And / or, the gain value is 2-16.

[0034] Preferably, in the HPLC-ELSD detection step, the evaporative light scattering detector has a drift tube temperature of 75°C and a carrier gas flow rate of 2.0 L / min.

[0035] Specifically, the method for detecting the D-glycophosphocholine content in L-α-glycophosphocholine involves dissolving the sample in anhydrous ethanol (containing 5% v / v isopropanol and 5% v / v methanol), filtering it through a microporous membrane (0.45 μm), accurately measuring the L-α-glycophosphocholine test solution, injecting it into a high-performance liquid chromatograph, acquiring and recording the chromatogram under the set instrument parameters, and substituting the peak area of ​​D-glycophosphocholine into the fitted linear curve of the L-α-glycophosphocholine reference standard series solutions. The content of D-glycophosphocholine isomer impurities is then quantitatively calculated using the L-α-glycophosphocholine external standard.

[0036] Specifically, in the analytical column, the packing material of the cellulose-type chiral normal phase chromatography column includes one of cellulose-tris(3-chloro-4-methylphenylcarbamate), cellulose-tris(3-chloro-5-methylphenylcarbamate), or cellulose-tris(3,5-dimethylphenylcarbamate).

[0037] In some specific embodiments, the selected chromatographic column is a 5μm, 250mm×4.6mm CHIRALPAKIM silica gel covalently bonded cellulose chiral normal phase chromatographic column.

[0038] Specifically, the method for detecting the D-glucosamine content in L-α-glucosamine choline includes the following method for preparing the L-α-glucosamine choline test solution: take the L-α-glucosamine choline test sample, accurately add a diluent to dissolve it, and filter it to obtain a solution with a concentration of 7.5-15 mg / ml.

[0039] Specifically, the detection method further includes the step of preparing an L-α-glucosinolate reference solution;

[0040] The steps for preparing L-α-glucosamine choline reference solution include: taking L-α-glucosamine choline reference standard, accurately adding diluent to dissolve it, and filtering to prepare a series of reference solution with concentrations of 3-15 μg / ml, such as 3.75, 7.5, 15 μg / ml and other gradient concentrations.

[0041] Specifically, the detection method further includes the step of preparing a system suitability solution;

[0042] The steps for preparing the system suitability solution include: taking L-α-glucosinolate and D-glucosinolate separately, accurately adding diluent to dissolve them, and filtering to prepare a mixed solution containing 5-15 mg / ml of L-α-glucosinolate and 3-6 μg / ml of D-glucosinolate.

[0043] Specifically, the diluent includes anhydrous ethanol or anhydrous ethanol containing both 3-8% v / v methanol and 3-8% v / v isopropanol.

[0044] Preferably, the diluent is anhydrous ethanol (containing 5% v / v isopropanol and 5% v / v methanol).

[0045] Secondly, the present invention provides the application of the method for detecting the D-glucosine content in L-α-glucosine in the field of L-α-glucosine quality analysis and detection.

[0046] The beneficial effects of this invention are as follows: The method for detecting D-glycophosphocholine content in L-α-glycophosphocholine described in this invention uses a normal-phase chromatographic column with covalently bonded cellulose-type or polysaccharide-coated cellulose-type silica gel and an evaporative light scattering detector (ELSD). Detection is performed using HPLC-ELSD (High Performance Liquid Chromatography-Evaporative Light Scattering Detection). Utilizing the cellulose-type chiral stationary phase covalently bonded to silica gel, in normal-phase chromatographic mode, differential retention and separation of enantiomers are achieved through multiple non-covalent interactions combined with steric hindrance. This method can efficiently separate and accurately quantify low-limit D-glycophosphocholine isomer impurities in the L-α-glycophosphocholine test sample. The content of low-limit D-glycophosphocholine isomer impurities in the test sample is calculated using an external standard of L-α-glycophosphocholine reference. This invention provides a high-performance liquid chromatography (HPLC) method that is simple to operate, highly sensitive, has good method specificity and reproducibility, and is easy to standardize. It can completely separate L-α-glucosinolate from low-limit D-glucosinolate isomer impurities and accurately quantify them.

[0047] The method for detecting the D-glucosamine content in L-α-glucosamine choline described in this invention employs HPLC (isocratic elution, evaporative light scattering detector) for detection. The mobile phase is a mixture of amine-containing alkanes and alcohols, which enables rapid and accurate determination of the low limit of D-glucosamine isomer impurities in L-α-glucosamine choline.

[0048] The method for detecting D-glucosamine content in L-α-glucosamine choline described in this invention employs an evaporative light scattering detector, which is highly sensitive, low-cost, and readily available, thus achieving effective control of low-limit D-glucosamine isomer impurities in high-purity L-α-glucosamine choline.

[0049] The method for detecting D-glucosamine content in L-α-glucosamine choline described in this invention uses mobile phase A:mobile phase B = 7:3 isocratic elution, avoiding the use of water as the mobile phase and dual-pump high-pressure gradient elution in liquid chromatography. This method results in less wear on the chiral column, extends its service life, and significantly reduces detection costs. Furthermore, the addition of an appropriate amount of amine to the mobile phase maintains the pH of the chromatographic system at approximately 6.2, preventing damage to the normal-phase chiral column from high acidity or alkalinity and further extending the column's lifespan.

[0050] The method for detecting D-glycophosphocholine content in L-α-glycophosphocholine described in this invention effectively overcomes the shortcomings of techniques that control the content of D-glycophosphocholine isomer impurities in L-α-glycophosphocholine through different methods. It has the advantages of simple operation, readily available reagents, and high separation. It can quickly and accurately quantify the content of D-glycophosphocholine isomer impurities in the test sample using the external standard method of L-α-glycophosphocholine. It has the advantages of high accuracy, sensitivity, and reproducibility, and the detection cost is greatly reduced.

[0051] Compared to traditional NMR phosphorus spectroscopy or high-performance liquid chromatography-differential detector methods, the analytical method of this invention has high sensitivity, low detection cost, and readily available solvents and detectors. It can conveniently and rapidly quantify the content of D-glycophosphocholine isomer impurities in L-α-glycophosphocholine, and can be applied to the quality control of L-α-glycophosphocholine products in industrialization. It overcomes the shortcomings of existing methods, such as the lack of a chromogenic group in D-glycophosphocholine, which prevents the use of ultraviolet detectors, low sensitivity of differential detectors, short lifespan of single normal-phase chiral chromatographic columns for isomer detection, and high cost of mass spectrometry. Attached Figure Description

[0052] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein:

[0053] Figure 1 This is the HPLC-ELSD spectrum of the solvent of this invention;

[0054] Figure 2 This is the HPLC-ELSD chromatogram of the solution suitable for the system of this invention;

[0055] Figure 3 This is the HPLC-ELSD chromatogram of the sensitive solution of this invention;

[0056] Figure 4 This is the HPLC-ELSD chromatogram of the reference solution of this invention;

[0057] Figure 5 This is the HPLC-ELSD chromatogram of the test solution of this invention;

[0058] Figure 6 This is a linear graph of L-α-glucosinolate from the present invention;

[0059] Figure 7 Chromatogram for the 0.65 ml / min robustness system suitability resolution;

[0060] Figure 8 Chromatogram for the 0.75 ml / min robustness system suitability resolution;

[0061] Figure 9 The detection spectrum is shown in Comparative Example 1. Detailed Implementation

[0062] The present invention provides a high-performance liquid chromatography (HPLC-ELSD) method for detecting the content of D-glycophosphocholine isomer impurities in L-α-glycophosphocholine in the following embodiments, namely, using HPLC-ELSD to detect the content of D-glycophosphocholine isomer impurities in L-α-glycophosphocholine. For detection, HPLC employed isocratic elution, using a chiral normal-phase column with cellulose covalently bonded to a silica gel surface or cellulose coated with a silica gel surface. Mobile phase A was an alkane solution containing 0.1-0.3% diethylamine or triethylamine, and mobile phase B was an alcohol solution containing 0.1-0.3% diethylamine or triethylamine. The volume ratio of mobile phase A to mobile phase B was (50-75):(50-25). The column temperature was 25℃-45℃, the flow rate was 0.5ml / min-1.0ml / min, and the injection volume was 20μl-50μl. An evaporative light scattering detector was used, with a detection drift tube temperature of 70℃-100℃ and a carrier gas flow rate of 1.5L / min-3L / min. The carrier gas could be high-purity nitrogen or air. The gain value G was 2-16.

[0063] In the high-performance liquid chromatography method for detecting the D-glucosine isomer impurity content in L-α-glucosine according to the present invention, the mobile phase is prepared using conventional methods in the art to obtain a mobile phase system that fully meets the analytical performance standards.

[0064] In the following embodiments of the present invention, the sample is dissolved in a selected diluent, such as anhydrous ethanol (99.8% by mass) or anhydrous ethanol (containing 5% v / v isopropanol and 5% v / v methanol), filtered through a 0.45 μm microporous membrane, and injected into a high-performance liquid chromatograph. Chromatograms are collected and recorded under the instrument parameters set, and D-glucosinolate is quantitatively calculated using the L-α-glucosinolate external standard method.

[0065] In the following embodiments of the present invention, before performing liquid phase detection, it is necessary to prepare L-α-glucosinolate reference curve solution, L-α-glucosinolate sensitivity solution, L-α-glucosinolate and limit concentration D-glucosinolate mixed solution as system adaptability solution, and L-α-glucosinolate test solution, respectively. The selected diluent is anhydrous ethanol containing 5% v / v isopropanol and 5% v / v methanol.

[0066] Preparation of L-α-glucosinolate test solution: Take an appropriate amount of L-α-glucosinolate, dissolve and dilute it with a solvent to prepare a solution containing 7.5 mg / ml to 15 mg / ml of L-α-glucosinolate, which is used as the L-α-glucosinolate test solution.

[0067] Preparation of L-α-glucosinolate reference solution: Take an appropriate amount of L-α-glucosinolate reference standard, dissolve it in a solvent, and dilute it to prepare a series of reference solutions with concentrations of 3 μg / ml to 15 μg / ml.

[0068] Preparation of L-α-glucosinolate sensitivity solution: Prepare L-α-glucosinolate reference solution at concentrations of 3 μg / ml to 6 μg / ml as sensitivity solution according to the above method for preparing reference solution.

[0069] Preparation of system suitability solution: Take an appropriate amount of D-glucosamine choline, dissolve and dilute it with diluent to prepare a stock solution containing 125 μg / ml to 250 μg / ml of D-glucosamine choline; take an appropriate amount of L-α-glucosamine choline, add an appropriate amount of solvent to dissolve it, accurately measure an appropriate amount of D-glucosamine choline reference stock solution, place it in the same volumetric flask, and dilute it with solvent to prepare a mixed solution containing L-α-glucosamine choline and D-glucosamine choline, which is used as the system suitability solution.

[0070] In the following embodiments of the present invention, 20 μl-50 μl each of the system suitability solution, the L-α-glucosinolate reference standard series solution, and the L-α-glucosinolate test solution are precisely measured and injected into the liquid chromatograph. The chromatograms are recorded, and the retention times of L-α-glucosinolate and D-glucosinolate in the chromatogram of the system suitability solution are marked. The resolution of the two substances is also recorded, and the resolution of the two substances should meet the requirements. For the L-α-glucosinolate reference standard series solution, a standard curve is plotted with the logarithm of the peak area as the abscissa and the logarithm of the injection mass as the ordinate. The linear fitting correlation coefficient r should be ≥0.99, and the signal-to-noise ratio (S / N) of the L-α-glucosinolate sensitivity solution should meet the requirements.

[0071] As an exemplary embodiment, in the following embodiments of the present invention, a CHIRALPAK IM normal-phase chiral chromatographic column with dimensions of 5 μm and a diameter of 250 × 4.6 mm is used as the analytical column. The CHIRALPAK IM normal-phase chiral chromatographic column uses cellulose-tris(3-chloro-4-methylphenylcarbamate) covalently bonded to silica gel as the chiral stationary phase. In normal-phase chromatographic mode, differential retention and separation of enantiomers are achieved through multiple non-covalent interactions combined with steric hindrance.

[0072] As an example, in the following embodiment of the present invention, the mobile phase A is a hexane solution containing 0.1% diethylamine, and the mobile phase B is an anhydrous ethanol solution containing 0.1% diethylamine (containing 5% v / v isopropanol and 5% v / v methanol).

[0073] As an example, in the following embodiments of the present invention, the flow rate of the mobile phase is 0.7 ml / min and the column temperature is 30°C.

[0074] As an example, in the following embodiment of the present invention, the drift tube temperature of the evaporative light scattering detector is 75°C, the carrier gas flow rate is 2.0 L / min, and the gain value is 2.

[0075] As an example, the present invention relates to the preparation methods of test solution, reference solution and system suitability solution in the following embodiments:

[0076] Preparation of test solution: Take an appropriate amount of L-α-glucosinolate test sample and dilute it with anhydrous ethanol (containing 5% v / v isopropanol and 5% v / v methanol) to prepare a test solution containing 7.5 mg / ml of L-α-glucosinolate.

[0077] Preparation of reference solutions: Take L-α-glucosinolate reference standard and dissolve and dilute it with anhydrous ethanol (containing 5% v / v isopropanol and 5% v / v methanol) to prepare reference standard curve solutions containing 3.75 μl / ml, 6.0 μl / ml, 7.5 μl / ml, 11.5 μl / ml, and 15 μl / ml of L-α-glucosinolate.

[0078] System suitability solution: Take appropriate amounts of L-α-glucosinolate and D-glucosinolate, dissolve and dilute them with anhydrous ethanol (containing 5% v / v isopropanol and 5% v / v methanol) to prepare a mixed solution containing 7.5 mg / ml of L-α-glucosinolate and 3.75 μg / ml of D-glucosinolate, which is the system suitability solution.

[0079] As an exemplary embodiment, in the following example of the present invention, 30 μl each of the system suitability solution and the L-α-glucosinolate standard curve solution are injected into the liquid chromatograph, the chromatograms are recorded, the retention times of L-α-glucosinolate and D-glucosinolate are marked, and the resolution of the two substances is recorded. The resolution should meet the requirements. The L-α-glucosinolate standard curve is recorded, and a standard linearity is plotted with the logarithm of the peak area as the abscissa and the logarithm of the injection mass as the ordinate. The correlation coefficient r should be ≥0.99.

[0080] The present invention will be further described below with reference to the embodiments and accompanying drawings.

[0081] Example 1

[0082] This embodiment provides a high-performance liquid chromatography (HPLC) method for the analysis of the D-glycophosphocholine isomer impurity content in L-α-glycophosphocholine. An HPLC instrument is used, and the specific chromatographic conditions are as follows:

[0083] Column: CHIRALPAK ® IM 250×4.6mm 5μm;

[0084] Column temperature: 30℃;

[0085] Flow rate: 0.7 ml / min;

[0086] Injection volume: 30 μl;

[0087] Evaporative light scattering detector (ELSD6000), drift tube temperature 75℃; carrier gas flow rate 2.0 L / min; gain value 2;

[0088] Mobile phase A: a hexane solution containing 0.1% v / v diethylamine;

[0089] Mobile phase B: Anhydrous ethanol containing 0.1% v / v diethylamine (containing 5% v / v isopropanol and 5% v / v methanol).

[0090] The mobile phase A:mobile phase B was eluted at a volume ratio of 7:3 (v / v).

[0091] In this embodiment, the diluent used for solution preparation is anhydrous ethanol (containing 5% v / v isopropanol and 5% v / v methanol).

[0092] Test solution: Accurately weigh L-α-glucosinolate, dissolve and dilute with diluent to prepare a solution containing 7.5 mg / ml of L-α-glucosinolate, which is used as the test solution.

[0093] Reference solutions: Accurately weigh an appropriate amount of L-α-glucosinolate reference standard, dissolve and dilute it with diluent to prepare a series of solutions containing 3.75 μl / ml, 6.0 μl / ml, 7.5 μl / ml, 11.5 μl / ml, and 15 μl / ml of L-α-glucosinolate as reference solutions. Inject the solutions in parallel and plot a standard curve.

[0094] System suitability solution: Weigh appropriate amounts of L-α-glucosinolate and D-glucosinolate, dissolve and dilute with diluent to prepare a mixed solution containing 7.5 mg / ml of L-α-glucosinolate and 3.75 μg / ml of D-glucosinolate, which is used as the system suitability solution.

[0095] Sensitivity solution: Take the lowest concentration (3.75 μl / ml) solution of L-α-glucosinolate reference standard.

[0096] Under the aforementioned chromatographic conditions, precisely measured 30 μL each of the system adaptability solution, reference solution, and test solution were injected into the liquid chromatograph, and the chromatograms were recorded. The content of D-glycine choline was calculated using the external standard method, with the logarithm of the peak area and the logarithm of the injected mass as the external standard.

[0097] In this embodiment, the content of D-glycine isomer impurities is calculated by peak area using the external standard method. The calculation formula is as follows:

[0098] ;

[0099] In the formula:

[0100] a: The slope of the standard curve for L-α-glucosamine choline reference solution;

[0101] b: Intercept of the standard curve for L-α-glucosinolate reference solution;

[0102] A: To determine the peak area of ​​the D-glucosamine isomer impurity in the test solution;

[0103] V: Dilution volume during preparation of the test solution, in ml;

[0104] c: Injection volume, μl;

[0105] m: The sample weight during the preparation of the test solution, in grams.

[0106] In this embodiment, the high-performance liquid chromatography system adaptability test and specificity test are carried out according to the above method.

[0107] The L-α-glucosamine choline reference solution was repeatedly injected to determine the correlation coefficient r, slope, and intercept of the standard curve; the lowest concentration reference solution was used as the sensitivity solution for detection once to determine the sensitivity of the evaporative light scattering detector; and a systematic solution injection was performed once to determine the separation effect between L-α-glucosamine choline and D-glucosamine choline.

[0108] Based on the properties of the substances, chromatographically pure anhydrous ethanol (containing 5% v / v isopropanol and 5% v / v methanol), L-α-glucosinolate test solution, L-α-glucosinolate and D-glucosinolate mixed system adaptability solution, and L-α-glucosinolate reference solution were injected separately.

[0109] The experimental results are attached. Figure 1-5 As shown in the figure. The overload peak is L-α-glycine, the subsequent smaller peak is D-glycine, and the peak before the retention time of 5 min is the solvent peak.

[0110] like Figure 1 The results show that the blank solvent, anhydrous ethanol (containing 5% v / v isopropanol and 5% v / v methanol), does not interfere with the detection of D-glucosinolate in L-α-glucosinolate.

[0111] like Figure 2 The results show that the main peak of L-α-glycine is completely separated from the adjacent impurity peaks and the impurity peak of D-glycine, with a resolution of 3.2. Other impurities do not interfere with the detection of D-glycine.

[0112] like Figure 3 The results show that the signal-to-noise ratio (S / N) of the evaporative light scattering detector is 10.7, which meets the sensitivity requirements.

[0113] like Figure 4 The results show that, taking the 6.0 μl / ml reference solution as an example, the relative standard deviation of the reference solution after 5 consecutive injections is 3.2%, indicating that the detection system is stable and can meet the detection requirements.

[0114] like Figure 5 The results show that D-glucosamine in L-α-glucosamine choline can be detected and accurately quantified.

[0115] As can be seen, in the detection method of this invention, chromatographically pure anhydrous ethanol (containing 5% v / v isopropanol and 5% v / v methanol) as blank solvent showed no interference peaks at the retention time position of the D-glucosinolate target analyte; in the L-α-glucosinolate and D-glucosinolate system-adaptive solutions, the two substances were completely separated; the retention time of the D-glucosinolate peak in the L-α-glucosinolate test solution was consistent with the retention time of the target impurity peak in the D-glucosinolate reference solution, and the resolution between the target impurity peak D-glucosinolate and adjacent impurity peaks was ≥1.5, indicating good resolution.

[0116] Example 2

[0117] This embodiment uses the above method to detect the limit of detection and the limit of quantitation.

[0118] Stock solution: Weigh 25.40 mg of L-α-glucosinolate reference standard, place it in a 100 ml volumetric flask, add chromatographic grade anhydrous ethanol (containing 5% v / v isopropanol and 5% v / v methanol) to dissolve and dilute to the mark, shake well, and this is the L-α-glucosinolate reference standard stock solution.

[0119] Limit of Quantitation Solution: Accurately transfer 1.5 ml of L-α-glucosinolate reference stock solution into a 100 ml volumetric flask, dilute to the mark with anhydrous ethanol (containing 5% v / v isopropanol and 5% v / v methanol), and the solution is ready.

[0120] Detection limit solution: Accurately transfer 1 ml of L-α-glucosinolate stock solution into a 100 ml volumetric flask, dilute to the mark with anhydrous ethanol (containing 5% v / v isopropanol and 5% v / v methanol), and the solution is ready.

[0121] Detection limit determination results: The detection solution concentration was 2.54 μg / ml, and the results are shown in Table 1. It can be seen that the signal-to-noise ratio (S / N) of all three consecutive injections was ≥3.

[0122] Table 1 Results of detection limit determination

[0123]

[0124] Quantitation limit determination results: The quantitation limit concentration was 3.81 μg / ml, and the detection results are shown in Table 2. It can be seen that when the test solution concentration was 3.81 μg / ml, after six repeated injections, the S / N ratio was greater than 10, indicating accurate quantification.

[0125] Table 2 Results of Limit of Quantitation Determination

[0126]

[0127] Example 3

[0128] This embodiment conducts linearity and range tests based on the above method.

[0129] Linear stock solution: Weigh 26.81 mg of L-α-glucosinolate reference standard, place it in a 100 ml volumetric flask, dissolve and dilute to the mark with anhydrous ethanol (containing 5% v / v isopropanol and 5% v / v methanol), shake well, and this is the stock solution.

[0130] The linear solutions at various concentration levels are shown in Table 3.

[0131] Table 3 Preparation of Horizontal Linear Solutions

[0132]

[0133] The test results are shown in Table 4 and Appendix 4 below. Figure 6 As shown.

[0134] Table 4 Linearity Test Results

[0135]

[0136] As can be seen, the fitted linear equation Y = 0.7652X - 2.3256 has a correlation coefficient (r) of 0.991. Within the concentration range of 2.6810 μg / ml to 8.0430 μg / ml, the logarithm of the peak area of ​​L-α-glycine phosphoric acid exhibits a good linear relationship with the logarithm of the injection mass, meaning there is a good linear relationship between the peak area and the concentration.

[0137] It is evident that D-glucosinolate is a chiral isomer of L-α-phosphocholine. The two substances have similar polarities, with correction factors ranging from 0.9 to 1.1. L-α-phosphocholine is readily available, and its limits of quantitation, detection, and linearity can be used to determine the limits of quantitation, detection, and linearity of D-glucosinolate. The content of D-glucosinolate isomer impurities can be quantitatively calculated using an external standard of L-α-phosphocholine.

[0138] Example 4

[0139] This embodiment conducts a recovery rate (accuracy) test based on the above method.

[0140] L-α-glucosinolate test solution: Weigh approximately 75 mg of L-α-glucosinolate accurately, place it in a 10 ml volumetric flask, dissolve and dilute to the mark with anhydrous ethanol (containing 5% v / v isopropanol and 5% v / v methanol) to obtain the background test solution.

[0141] L-α-glucosinolate standard curve solution: Weigh 26.81 mg of L-α-glucosinolate and place it in a 100 ml volumetric flask. Take 1200 μl, 1500 μl, 1800 μl, 2400 μl, and 3000 μl of each solution and place them in separate 100 ml volumetric flasks. Add anhydrous ethanol (containing 5% v / v isopropanol and 5% v / v methanol) to make up to volume to prepare the standard curve solution.

[0142] D-glucosamine choline stock solution: Weigh 12.67 mg of D-glucosamine choline reference standard, place it in a 100 ml volumetric flask, add anhydrous ethanol (containing 5% v / v isopropanol and 5% v / v methanol) to dissolve and dilute to the mark, shake well, and the D-glucosamine choline reference standard stock solution is obtained.

[0143] Spiked recovery solutions: Weigh approximately 75 mg of L-α-glucosinolate sample into 9 portions and prepare mixed solutions of low, medium, and high concentrations of spiked test sample, respectively, to obtain spiked test sample solutions with recovery rates of 80%, 100%, and 120%. Prepare 3 portions for each concentration level and determine the recovery rate according to the detection method. The results are shown in Table 5.

[0144] Table 5 Spike Recovery Test Results

[0145]

[0146] As can be seen, the average recovery rate of the lower limit D-glucosinolate at the three concentrations was 108.8%, ranging from 80% to 120%, and the RSD values ​​of the recovery rates at each concentration were 4.8% and ≤10.0%, confirming that the method has good recovery rate (accuracy) for the detection of the lower limit D-glucosinolate in L-α-glucosinolate.

[0147] Example 5

[0148] This embodiment is based on the above method to conduct repeatability tests.

[0149] Six spiked test solutions at medium concentration levels were prepared as repeatability solutions, and the results are shown in Table 6 below.

[0150] Table 6 Repeatability Test Results

[0151]

[0152] As can be seen, in the six repeated injections of the spiked test solution, the RSD value of D-glucosinolate in the spiked test solution was 0.8%, indicating that the method has good repeatability.

[0153] Example 6

[0154] This embodiment conducts a solution stability experiment based on the above method.

[0155] D-glucosamine choline reference solution: Weigh 12.67 mg of D-glucosamine choline reference standard, place it in a 100 ml volumetric flask, add anhydrous ethanol (containing 5% v / v isopropanol and 5% v / v methanol) to dissolve and dilute to the mark, shake well, and use as the D-glucosamine choline reference standard stock solution.

[0156] Medium-concentration spiked test solution: Weigh 76.78 mg of L-α-glucosinolate accurately and place it in a 10 ml volumetric flask. Add an appropriate amount of anhydrous ethanol (containing 5% v / v isopropanol and 5% v / v methanol) to dissolve it. Measure 300 μl of D-glucosinolate reference stock solution and place it in the same 10 ml volumetric flask. Dilute to the mark with anhydrous ethanol (containing 5% v / v isopropanol and 5% v / v methanol).

[0157] The sample was placed at room temperature and injected at 0:00, 8:00 and 12:00 respectively. The changes in the target impurity peak were calculated, and the results are shown in Table 7 below.

[0158] Table 7 Solution stability results

[0159]

[0160] It can be seen that the peak area of ​​D-glucosamine impurities at each time point was between 80% and 120% relative to the peak area of ​​D-glucosamine impurities at time 0, confirming that D-glucosamine in L-α-glucosamine solution has good stability within 12 hours.

[0161] Example 7

[0162] This embodiment examines the durability of the method based on the above method.

[0163] Under the chromatographic conditions defined in Example 1 above, the flow rate was fine-tuned, and the system suitability resolution at the adjusted flow rate (±0.05 ml / min) was specifically investigated. The results are shown in Table 8 below. The robustness system suitability resolution chromatogram at 0.65 ml / min is attached. Figure 7 As shown in the attached diagram, the system's robustness and suitability for resolving chromatograms at a flow rate of 0.75 ml / min are suitable for various applications. Figure 8 As shown.

[0164] Table 8 Results of Durability System Suitability Tests

[0165]

[0166] As can be seen, the calculated results of the D-phosphocholine isomer impurity content in the sample are all within the range required by the verification scheme. The system adaptability meets the requirements when the flow rate is changed. The RSD of the D-phosphocholine isomer impurity content under different durability conditions is 3.7% (the specified value is ≤10.0%), which confirms that the invention method has good durability.

[0167] Comparative Example 1

[0168] This comparative example compares the composition of the mobile phase during isocratic elution under the chromatographic conditions specified in Example 1 above.

[0169] In this comparative example, mobile phase A was a hexane solution and mobile phase B was anhydrous ethanol (99.8% by mass). The process involved online mixing and isocratic elution with dual pumps and an evaporative light scattering detector. The elution run time was 70 minutes, and the mobile phase ratio was 70:30 (v / v) for mobile phase A and mobile phase B.

[0170] As attached Figure 9 The results show that under these chromatographic conditions, the peak of L-α-glycine in the test solution exhibited severe tailing, interfering with the detection of the target D-glycine isomer impurity. The two substances could not be completely separated, and D-glycine could not be accurately quantified.

[0171] Comparative Example 2

[0172] In this comparative example, under the chromatographic conditions specified in Example 1 above, a differential detector was used instead of an evaporative light scattering detector for detection.

[0173] In this comparative example, cellulose-tris(3-chloro-4-methylphenylcarbamate) covalently bonded to the surface of silica gel was used. ® The mobile phase consisted of a 4.6 mm × 250 mm 5 μm IM filter, a 70:30 volume ratio of a solution containing 0.1% diethylamine-n-hexane and 0.1% diethylamine-anhydrous ethanol (containing 5% v / v isopropanol and 5% v / v methanol), premixed and eluted isocratically, and detected by a differential detector when the concentration of the L-α-glucosinolate test sample solution was 15 mg / ml.

[0174] Under these detection conditions, due to the lack of UV absorption by the main components and the limited sensitivity of the differential detector, L-α-glucosinolate and D-glucosinolate cannot be completely separated, and D-glucosinolate cannot be detected at the low limit (0.05%) of L-α-glucosinolate, thus the detection sensitivity does not meet the detection requirements.

[0175] As can be seen, compared with the traditional high performance liquid chromatography-differential detector detection method, the analytical method of this invention has advantages such as high sensitivity, low detection cost, and easy availability of solvents and detectors. It can conveniently and quickly quantify the content of D-glucosinolate isomer impurities in L-α-glucosinolate, and overcome the shortcomings of existing D-glucosinolate detection methods, which have no chromogenic group and cannot be used with ultraviolet detectors, have low sensitivity of differential detectors, and have short lifespan of single normal-phase chiral chromatographic columns for isomer detection.

[0176] In summary, the detection method described in this invention has high accuracy, precision, and sensitivity, and is simple and convenient to operate. It can achieve rapid and accurate quantification of low-limit D-glucosamine isomer impurities in high-purity L-α-glucosamine choline, effectively saving detection time and costs.

[0177] The embodiments of the present invention have been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of the present invention. The descriptions of the embodiments above are only for helping to understand the method and core ideas of the present invention. At the same time, for those skilled in the art, there will be changes in the specific implementation methods and application scope based on the ideas of the present invention. Therefore, the content of this specification should not be construed as a limitation of the present invention.

Claims

1. A method for detecting the content of D-glycine in L-α-glycine, characterized in that, The detection method includes the step of performing HPLC-ELSD detection on the L-α-glucosinolate test solution; The chromatographic conditions in the HPLC-ELSD detection step include: The analytical column was a chiral normal-phase column of cellulose covalently bonded to a silica gel surface with polysaccharides or a chiral normal-phase column of cellulose coated with silica gel with polysaccharides. The mobile phase A is an alkane solution containing 0.1-0.3% v / v diethylamine or triethylamine; Mobile phase B is an alcoholic solution containing 0.1-0.3% v / v diethylamine or triethylamine; The mobile phase A and mobile phase B are eluted isocratically at a volume ratio of (50-75):(50-25).

2. The method for detecting the content of D-glucosamine in L-α-glucosamine according to claim 1, characterized in that: In the mobile phase A, the alkanes in the alkane solution include n-hexane, n-heptane, or isooctane; and / or, In the mobile phase B, the alcohol in the alcohol solution includes isopropanol, anhydrous ethanol, or anhydrous ethanol containing both 3-8% v / v methanol and 3-8% v / v isopropanol.

3. The method for detecting the content of D-glycine in L-α-glycine according to claim 1, characterized in that, The chromatographic conditions in the HPLC-ELSD detection step also include: The flow rate is 0.5-1.0 ml / min; and / or, Column temperature is 25-45℃; and / or, The injection volume is 20-50 μl; and / or, The detection time is 40-60 minutes.

4. The method for detecting the content of D-glycine in L-α-glycine according to claim 1, characterized in that, In the HPLC-ELSD detection step, the settings parameters of the evaporative light scattering detector include: The drift tube temperature is 70-100℃; and / or, The carrier gas includes high-purity nitrogen or air; and / or, The carrier gas flow rate is 1.5-3.0 L / min.

5. The method for detecting the content of D-glycine in L-α-glycine according to claim 1, characterized in that, In the analytical column, the packing material of the cellulose-type chiral normal phase chromatographic column includes one of cellulose-tris(3-chloro-4-methylphenylcarbamate), cellulose-tris(3-chloro-5-methylphenylcarbamate), or cellulose-tris(3,5-dimethylphenylcarbamate).

6. The method for detecting the content of D-glucosamine in L-α-glucosamine according to claim 1, characterized in that, The preparation method of the L-α-glucosinolate test sample solution includes: taking L-α-glucosinolate test sample, accurately adding diluent to dissolve it, and filtering to obtain a solution with a concentration of 7.5-15 mg / ml.

7. The method for detecting the content of D-glycine in L-α-glycine according to claim 1, characterized in that, The detection method further includes the step of preparing an L-α-glucosinolate reference solution; The steps for preparing L-α-glucosinolate reference solution include: taking L-α-glucosinolate reference standard, accurately adding diluent to dissolve it, and filtering to prepare a series of reference solutions with a concentration of 3-15 μg / ml.

8. The method for detecting the content of D-glycine in L-α-glycine according to claim 1, characterized in that, The detection method further includes the step of preparing a system suitability solution; The steps for preparing the system suitability solution include: taking L-α-glucosinolate and D-glucosinolate separately, accurately adding diluent to dissolve them, and filtering to prepare a mixed solution containing 5-15 mg / ml of L-α-glucosinolate and 3-6 μg / ml of D-glucosinolate.

9. The method for detecting the content of D-glycine in L-α-glycine according to any one of claims 6-8, characterized in that, The diluent includes anhydrous ethanol or anhydrous ethanol containing both 3-8% v / v methanol and 3-8% v / v isopropanol.

10. The application of the method for detecting the D-glucosamine content in L-α-glucosamine according to any one of claims 1-8 in the field of L-α-glucosamine quality analysis and detection.

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